Efficient production of bispecific antibodies-optimization of transfection strategy leads to high-level stable cell line generation of a Fabs-in-tandem immunoglobin.

Bispecific antibodies (bsAbs) are often composed of more than two component chains, such as Fabs-in-tandem immunoglobin (FIT-Ig) comprising three different component chains, which bring challenges for generating a high proportion of the correctly assembled bsAbs in a stable cell line. During the CHO-K1 stable cell line construction of a FIT-Ig, we investigated the FIT-Ig component chain ratio in transfection, where two sets of expression vectors were designed. Both designs utilized two vectors for co-transfection. Multiple transfections with plasmid ratio adjustment were applied, and the resultant minipools were evaluated for expression titer and quality of produced FIT-Ig. The results suggested that abundant outer Fab short chains (twofold chain genes versus other chains) can promote complete FIT-Ig assembly and therefore reduce the fragmental impurities of FIT-Ig. This adjustment of the component chain ratios at the beginning is beneficial to FIT-Ig stable cell line generation and brings favorable clones to process development.

Generation of Fabs-in-tandem immunoglobulin molecules for dual-specific targeting.

Bispecific antibody (BsAb) has become an important trend in developing next generation biologics therapies. By simultaneously engaging two molecular targets, BsAbs show distinctive mechanism of actions that could lead to clinical benefits unattainable by conventional monoclonal antibodies (mAbs). Successful launch provided clinical validation and encourage more BsAb development in the pipeline of pharmaceutical companies. Fabs-in-tandem immunoglobulin (FIT-Ig™) format was initially described in 2017. This unique design provides a symmetrical and tetravalent IgG-like bispecific molecule with correct association of 2 sets of VH/VL pairs, where two Fabs are fused directly in a crisscross orientation without any mutations or use of peptide linkers. FIT-Ig can be readily made from 2 existing monoclonal antibodies by basic molecular biology techniques with high expression level in mammalian cells, and easily purified to homogeneity using standard approaches without extensive optimization. FIT-Ig molecules exhibit favorable drug-like properties, in vitro and in vivo functions, as well as manufacturing efficiency for commercial development. Here, we provide an example of construction and preliminary characterization of a FIT-Ig molecule with discussions on optimization and general utility.

Fabs-in-tandem immunoglobulin is a novel and versatile bispecific design for engaging multiple therapeutic targets.

In recent years, the development of bispecific antibody (bsAb) has become a major trend in the biopharmaceutical industry. By simultaneously engaging 2 molcular targets, bsAbs show unique mechanisms of action that could lead to clinical benefits unattainable by conventional monoclonal antibodies. Various bsAb generation formats have been described, and several are being investigated in clinical development. However, some bsAb constructs have proven to be problematic due to their unfavorable physicochemical and pharmacokinetic properties, as well as poor manufacturing efficiencies. We describe here a new bispecific design, Fabs-in-tandem immunoglobulin (FIT-Ig), in which 2 antigen-binding fragments are fused directly in a crisscross orientation without any mutations or use of peptide linkers. This unique design provides a symmetric IgG-like bispecific molecule with correct association of 2 sets of VH/VL pairs. We show that FIT-Ig molecules exhibit favorable drug-like properties, in vitro and in vivo functions, as well as manufacturing efficiency for commercial development.